PROJECT SUMMARY/ABSTRACT Spermatozoa acquire their ability to fertilize an oocyte as they transit through the epididymis. The luminal fluid of the epididymal duct is acidic and has a low bicarbonate (HCO3-) concentration: both factors are important for keeping sperm quiescent during their maturation and storage. In the previous funding periods, we showed that several population of epithelial cells that line the epididymal lumen work in a concerted manner to regulate luminal pH. Clear cells (CCs) acidify the lumen via the vacuolar proton pump V-ATPase located in their apical membrane. During sexual arousal, stimulated principal cells (PCs) secrete HCO3- into the lumen, which contributes to luminal alkalinization, a process that primes sperm before ejaculation. We showed that, in addition to HCO3-, PCs also secrete adenosine triphosphate (ATP) into the lumen. Male infertility is associated with a defect in purinergic regulation by ATP and its hydrolysis product adenosine. Our previous data revealed that PCs and CCs engage in crosstalk that relies not only on HCO3-, but also on ATP and adenosine, which then act as extracellular mediators that activate H+ secretion by CCs to restore luminal pH to its resting acidic condition. Importantly, we also found that in the steady state, PCs contribute to luminal acidification via the Na+/H+ exchanger NHE3. PCs can, therefore, switch from being acid-secretors to base-secretors in response to physiological cues. Aim 1 will characterize this dual pH-regulating function of PCs by answering the question: how do PCs switch from being H+-secreting to HCO3--secreting cells? We will examine the role of circulating agonists in this switch between H+ secretion and HCO3- secretion by PCs. We will also characterize ATP secretion by PCs, and its hydrolysis via extracellular ectonucleotidases to produce adenosine. Aim 2 will define the PC-CC crosstalk that regulates H+ secretion by CCs. We will dissect the purinergic regulation of V-ATPase- dependent H secretion by CCs. We will use two-photon intravital microscopy to characterize the role of HCO3- + , ATP and adenosine as extracellular mediators that are secreted from PCs to regulate V-ATPase-dependent H+ secretion in CCs. Our proposed multidisciplinary approach involves a battery of complementary and innovative in vivo and in vitro procedures. These studies will provide new insights into the regulation of luminal acidification in the epididymis, and will dissect intercellular communication pathways that are essential for the establishment of male fertility. Ultimately, a better understanding of basic mechanisms involved in sperm maturation in the post-testicular male reproductive tract will drive clinical advances for the treatment of male infertility.